Recombinant expression systems have been key to the development of current antibody engineering technology. The demonstration of coexpression of cloned light and heavy chain genes of an IgM or an IgG in mammalian cells led rapidly to the generation and testing of chimeric Mabs containing human constant regions (Ochi et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:6351 6355; Oi et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:825 829; Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81(21):6851-5). Subsequently, methods were developed to introduce human sequences into the variable regions of mouse immunoglobulins without reducing avidity, resulting in antibodies with very low potential immunogenicity in human subjects (Jones et al., 1986, Nature 321:522 525; Queen et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:10029 10033). Reproducible methods have been developed to express large amounts of such recombinant antibodies in CHO or mouse myeloma cells for the preparation of highly purified material for human testing and eventual sale. There are now a number of such Mabs which have been approved and marketed for human use, including Rituxan and Herceptin for cancer treatment, Synagis for the prevention of RSV infection, Remicade for treatment of rhumatoid arthritis, and Zenapax for prevention of graft rejection (Reff et al., 1994, Blood 83:435 445; Carter et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89(10):4285-9; Johnson et al., 1997, J. Infect. Dis. 176:1215 1224; Queen et al., 1989, Proc. Natl. Acad. Sci. U.S.A 86:10029 10033; see Table 1).
Likewise, the demonstration that Fv, single chain Fv, or Fab molecules could be successfully expressed in microbial systems led rapidly to the development of methods to utilize this expression technology to exploit diverse libraries of VH and VL sequences (Skerra et al., 1988, Science 240:1038 1041; Bird et al., 1988; Science 242:423 426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A 85:5879 5883). Combinatorial libraries of VL and VH sequences were initially expressed from bacteriophage lambda and the binding of a particular combination to antigen screened using a plaque lift assay (Huse et al., 1989, Science 246:1275-81; Huse et al., 1992, Biotechnology 24:517 523). By tethering either an scFv or Fab on the surface of a filamentous bacteriophage it was possible to select for binding phage containing the genes for the binding regions in their genome using panning techniques (McCafferty et al., 1990, Nature 348:552 554; Hoogenboom et al., 1991, 19: 4133 4137; Bird et al., 1988, Science 242:423 426; Kang et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:4363 4366). The ability to select rather than screen allowed the enormous diversity contained in large libraries of 109 or greater individual members to be exploited to identify and isolate rare binders. Thus, it is now possible to isolate antibody fragments binding with reasonable affinity to almost any proteinaceous antigen from a large diverse bacteriophage library. Methods have also been developed to improve the affinity of antibody fragments by iterative rounds of mutagenesis of the CDRs and screening or selecting for improved binding to antigen (Schier et al., 1996, J. Mol. Biol. 263: 551 567; Wu et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:6037 6042).
Despite breakthroughs in using Fab or scFv expression systems for the identification and affinity maturation of novel specificities, full length IgG molecules offer several advantages. One of the key features of an IgG is its bivalent structure. Cooperativity between the two Fab arms of an IgG in binding to antigen leads to a higher avidity of a bivalent IgG compared to the monovalent Fab. The degree of difference between the affinity of the individual Fab arms and the avidity of the IgG is most pronounced when the antigen is also multivalent or surface bound. The amount of cooperativity is more pronounced when the antigen is present at higher density and less pronounced in Mab with high affinity Fab arms. In practical terms, this means that above a certain antigen density threshold a Mab with high affinity but low cooperativity will have the same avidity as a Mab having Fab arms with moderate affinity but high cooperativity. This latter Mab would be more selective for areas of high antigen density compared to the former Mab. One can envision instances where either Mab would be advantageous. For instance, there are very few true cancer antigens, i.e., antigens which are expressed only on tumor cells. Most are expressed on tumor cells at a higher density, but are expressed on other cell types as well. Thus, a Mab with high avidity but moderate affinity might be more selective for tumor than for normal cells expressing the antigen at a lower density. Likewise, during viral infection, antigen may be present on the virus, on virally infected cells, and secreted in free form. A neutralizing Mab selective for areas of higher antigen density could target the virus and infected cells rather than free antigen, or other areas of low antigen density, and thus might have equal or better efficacy compared to a high affinity Mab. Methods have been developed to select for higher avidity fragments using various strategies to link monomers of Fab or scFv (Hudson et al., 1999, J. Immunol. Methods 231:177 189). These constructs are useful but may not accurately replicate the avidity provided by linking Fab arms using an Fc. Additionally, one may want to first identify specific binders and then those which have higher cooperativity. For instance, in the example above, one might want to screen for viral neutralization but find that most monovalent Fabs had little activity. Converting to full length IgG might allow selection for neutralizing activity due to increased avidity.
In other cases, effector function may be required for optimal potency of the binding molecule. The interaction between the Fc portion of immunoglobulin molecules and specific cell surface receptors allows the coupling of antigen binding to effector cell functions.
There are three classes of Fc receptors for IgG present in humans and rodents, which are designated RI, RII, and RIII (Ravetch and Bolland, 2001, Annu. Rev. Immunol. 19: 275 290). RI, present on monocytes and macrophages, binds to monomeric IgG with high affinity. RII is present on a wide variety of cells including B cells, platelets, neutrophils, macrophages and monocytes, and binds to multimeric IgG (immune complexes or aggregated IgG) with moderate affinity. Two forms of RII are expressed, differing by the presence of either an activation (ITAM) RIIa domain or an inhibitory (ITIM) RIIb domain on the intracellular portion of the receptor. The relative level of activating and inhibitory receptors on a given cell determines the response to immune complexes. B cells express only the inhibitory form. RIII, like RII, binds to multimeric IgG (immune complexes or aggregated IgG) with moderate affinity. There are also two forms of RIII. The ITAM domain on the associated gamma chain mediates signaling through RI, as well as through RIIIa and the FcE receptors. The signaling molecule RIIIa associates with the ITAM containing gamma chain on NK, monocytes, macrophages, and certain T cells. On NK cells, signaling by RIIIa also involves the TCR zeta chain. RIIIb is a non signaling form and is expressed on (human) neutrophils as a GPI linked molecule.
In the body, RI sites are generally occupied by monomeric IgG while RII and RIII receptors are unoccupied and available to interact with immune complexes. Cross linking of activating Fc receptors by antibody antigen complexes can result in the phagocytosis of pathogens, killing of foreign and transformed cells by direct cytotoxicity, the clearance of toxic substances, and the initiation of an inflammatory response. Additionally, the Fc contains sites for interacting with complement components (Tao et al., 1993, J Exp Med 178:661 667). Finally, the Fc is responsible for the long half-life in vivo of IgGs through a specific interaction with the MHC related FcRn receptor (Ghetie and Ward, 2002, Immunol Res. 25:97 113).
Clearly, in instances where the target is a bacterium or a cancerous cell, it would be advantageous to test agents for clearance or killing rather than only binding. In that case, an IgG would be the preferred molecule to test. For instance, the chimeric anti CD20 Mab Rituxan was selected based on its having strong ADCC activity against human B cells (Reff et al., 1994, Blood 83:435 445). Additionally, although the anti HER2 antibody Herceptin binds to and blocks signaling through an EGF like receptor on tumor cells, recent studies have indicated that tumor protection is largely Fc-mediated (Clynes et al., 2000, Nat. Med. 6:443 446).
There is a great interest in expression, selection and improvement of antibodies using scFv or Fab systems. Using the technologies currently available, however, the resulting scFv or Fab fragments must be re-cloned into a vector for expression of the full length glycosylated Mab for further testing and development. This step severely limits the number of Mabs which can be tested at this stage. Thus, despite such interest in the technology, as yet, no effective system for selecting and improving full length Mab molecules useful for human therapeutics has been developed.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.